Central to the ability of vascular cells to adhere to the extracellular matrix and to each other is an abundant supply of cell surface adhesion molecules that, in addition to influencing the adhesive phenotype of the cell, are also capable of transmitting signals into, and responding to signals from, the cell interior. Such post-ligand events occur by virtue of the ability of these transmembrane proteins to interact with intracellula kinases and phosphatases, G-proteins, adapter proteins, and cytoskeletal components. PECAM-1 (also known as CD31) is a cellular adhesion and signaling receptor comprised of six extracellular immunoglobulin (Ig)-like homology domains, a short transmembrane domain, and a 118 amino acid cytoplasmic domain that becomes serine and tyrosine phosphorylated upon cellular activation. PECAM-1 expression is restricted to blood and vascular cells. In circulating platelets and leukocytes, PECAM-1 functions largely as an inhibitory receptor that, via regulated sequential phosphorylation of its cytoplasmic domain, limits cellular activation responses. PECAM-1 is also highly expressed at endothelial cell intercellular junctions, where it functions as a mechanosensor, as a regulator of leukocyte trafficking, and in the maintenance of endothelial cell junctional integrity. Previous studies have shown that PECAM-1-PECAM-1 homophilic interactions mediated by N-terminal Ig domain 1 are required for border localization, and contribute importantly not only to steady-state endothelial cell barrier stability, but also to recovery of endothelial cell junctional integrity, both in vitro and in vivo, following inflammator or hemostatic challenge. The overall goal of this competitive renewal application is to build on recent progress in the field of PECAM-1 biology to address outstanding, broadly applicable, and scientifically-important questions in the fields of cell adhesion and regulation. Two Specific Aims will focus on novel mechanisms that regulate the homophilic binding affinity of PECAM-1, while a third Specific Aim will deal with the cellular mechanisms of control over PECAM-1 phosphorylation that impose tight, temporal regulation of its inhibitory function. Specifically, ovr the next five-year period we propose to (1) Examine the structural and functional basis of allosteric regulation of PECAM-1 homophilic binding affinity, (2) Identify serine/threonine kinase(s) that trigger PECAM-1-mediated inhibitory signaling, and (3) Define atomic-level structural determinants of PECAM-1-mediated homophilic interactions that account for its ability to regulate and maintain endothelial cell junctional integrity. Together, these studies comprise a coordinated, focused research program designed to improve our understanding of the function of this novel vascular cell adhesion and signaling molecule in the blood and vascular cells in which it is expressed. Many of the principles established in this proposal, including affinity modulation within the Ig superfamily and the regulation of ITIM-mediated inhibitory signaling by sequential phosphorylation, should be impactful well beyond the field of PECAM-1 biology.